In recent years, enzyme immunoassay methods have received widespread acceptance in both research and commercial settings for use in the detection and measurement of antibodies or antigens of interest ("ligands") in test samples. Enzyme immunoassay methods involve the ultimate attachment of an enzyme label to a test sample ligand. The attachment may be either direct or indirect through enzyme-labeled immunocomplexes which bind specifically to the test sample antigen or antibody. The enzyme, in turn, catalyzes a reaction with a substrate to produce detectable signal.
The same general methods can be employed for the determination of ligands of interest other than antigens and antibodies. For example, where the ligand of interest is a piece of genetic material, such as a target nucleotide sequence, enzyme-labeled nucleic acid probes can be used to assay the target nucleotide of interest in a test sample. See, for example, U.S. Pat. No. 4,581,333, which is incorporated herein by reference.
Typical enzyme-labeled specific binding assays include both competitive and non-competitive, i.e. sandwich, type enzyme-labeled specific binding assays for ligands of interest. In a competitive enzyme-labeled specific binding assay method, a predetermined quantity of enzyme-labeled ligand competes with unlabeled test sample ligand for the available binding sites on a limited amount of insolubilized specific binding partner. The amount of insolubilized, or bound, enzyme-labeled ligand can be measured, with the amount of test sample ligand being determined by the relative proportion of specific binding partner--enzyme labelled ligand to specific binding partner--non-labelled ligand. The amount of ligand in the sample is indirectly proportional to the amount of enzyme-labelled ligand bound to the insolubilized specific binding partner. The specific binding partner can itself be bound to a second insolubilized binding partner.
In a non-competitive, or sandwich type specific binding assay, a first specific binding partner to the ligand of interest is insolubilized on a supporting membrane, particle, or similar material. A second specific binding partner to the ligand, preferably having a binding site(s) on the ligand different from that of the first specific binding partner, is labeled with an enzyme. The first and second specific binding partners will "sandwich" the ligand of interest, with the first insolubilized specific binding partner providing a means for separating the bound enzyme-labeled ligand from the free labeled ligand in solution. Alternatively, the first specific binding partner may be provided with a means for subsequent insolubilization after the desired sandwich complex is formed. The amount of labeled sandwich formed bears a direct relationship to the amount of ligand of interest present in the test sample.
The enzyme label in an enzyme-labeled specific binding assay provides a means of quantifying the amount of ligand present in a test sample once the desired labeled complex is formed. This is ordinarily accomplished by adding an excess of enzyme substrate to the solution containing the labeled complex. The substrate typically contains a chromogenic material which yields a colored product that can be conveniently detected both visually and instrumentally, such as with a spectrophotometer. The amount of ligand of interest in a test sample is directly or indirectly proportional to the "amount" of color produced by enzymatic action on the chromogenic material.
Where an excess of substrate is added, the rate of color change of the chromogen is independent of the substrate concentration, and the enzyme concentration becomes the rate-limiting factor in the overall color reaction. I.e., the rate of color change is a function of the enzyme concentration which is, in turn, a function of the amount of ligand of interest present in the test sample. This is important where measurement is accomplished by an instrument, because it allows for rapid quantification based on rate measurement, rather than the lengthy wait required to achieve a result in an end point method.
Typical enzymes that have been used in enzyme-labeled specific binding assays include peroxidase, glucose oxidase, .beta.-D-galactosidase, and alkaline phosphatase. The term "peroxidase" as used herein refers to any enzyme exhibiting oxidase or peroxidic activity and includes, for example, horseradish peroxidase, catalase, tyrosine oxidase and the like, Both .beta.-D-galactosidase and alkaline phosphatase are found in normal human urine; they are not ordinarily preferred for use in connection with enzyme-labeled specific binding assay techniques. Horseradish peroxidase, on the other hand, is not ordinarily found in human test samples. It is also plentiful, inexpensive, and stable, and has a high conversion rate of various chromogens in the presence of a peroxide to yield colored products.
The typical substrate for the enzyme horseradish peroxidase is a solution of a peroxide, such as hydrogen peroxide or urea peroxide, combined with a chromogenic material. Horseradish peroxidase first catalyzes the decomposition of the enzyme substrate peroxide. The degradation product then reacts with the chromogenic material to produce a chromophore which can be measured visually or spectrophotometrically. One of the most sensitive chromogenic materials available for detection of peroxidase activity is ortho-phenylenediamine (OPD), which is initially soluble and colorless in aqueous solution, but produces a yellowish-orange chromophore upon oxidation in the presence of a peroxide. Other chromogenic materials which are useful in the detection of peroxidase activity include: o-tolidine; 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); m-phenylenediamine; dianisidine; aniline; phenol red; pyrogallol; 4-aminoantipyrine; and bromopyrogallol red.
Previous OPD reagents are provided as one-part reagents with a substrate buffer being combined with the peroxide and chromogen. See generally, U.S. Pat. No. 4,234,680; U.S. Pat. No. 4,467,031; U.S. Pat. No. 4,444,879; U.S. Pat. No. 4,504,587; U.S. Pat. No. 4,520,113, as representative of the use of peroxide substrates and OPD chromophore, each of which is incorporated herein by reference. Commercially available OPD in a convenient tablet form may be obtained from a number of sources such as, for example, Beckman Instruments, Inc. (Product Number 688035--each tablet contains: 10 mg ortho-phenylenediamine plus mannitol, sodium carbonate, citric acid and polyethylene glycol).
One of the primary disadvantages of, for example, a peroxidase--OPD system is that the substrate can be oxidized by air to yield the same colored products. E.g., when a solution of OPD is dissolved in a peroxide and substrate buffer and left to stand alone, without the presence of a peroxidase enzyme, the OPD chromogen, in as little as one hour, rapidly oxidizes to a yellow-orange color. The air oxidation of the substrate can result in high blank values (defined as elevated absorbance values in the absence of enzyme activity), artificially high results, and other related problems. This, in turn, has an adverse effect on the reproducibility and precision of enzyme-labeled specific binding assays using a peroxidase--OPD system. To accommodate this problem, conventional chromogenic reagents are preferably prepared in fresh batches immediately prior to use. See, for example, U.S. Pat. No. 4,239,743, Col. 7, lines 12-18, and U.S. Pat. No. 4,503,143, Col. 3, lines 44-48, which are incorporated herein by reference.
This is a distinct disadvantage with enzyme immunoassays which themselves require multiple fluid transfers and manipulations, including addition of reactants, mixing, separation of solid and liquid phases, removal of unreacted components and undesired reaction products, washings, etc. These steps usually must be repeated over and over in order to achieve the desired end result; as such, these manipulations require a lot of time, which is at a premium in a clinical laboratory. Having to mix a fresh batch of chromogenic reagents for each assay thus exacerbates an already difficult problem and all but excludes the use of automated assay instruments that are designed to run such assays over extended time periods.
It would be advantageous to have a conventional substrate solution for peroxidase that exhibits improved stability over previous solutions, such that a fresh batch of reagent need not be prepared for each assay.